Carboxyl-terminal binding protein 1 (CtBP1), a well-known transcriptional co-repressor, is highly expressed in a number of cancer types. However, it is still absent in osteosarcoma cells. Here, we found that CtBP1, but not CtBP2, is overexpressed in invasive osteosarcoma tissues and cells. The overexpressed CtBP1 in turn represses its downstream targets, such as the pro-apoptotic regulators Bax, Bim and p53 upregulated modulator of apoptosis (PUMA), cell adhesion molecule E-cadherin, and the cell cycle regulators p16, p21 and phosphatase and tensin homolog (PTEN). To explore the molecular mechanism of CtBP1 overexpression, we subjected three independent clinical samples to miRNA microarray analysis and found that miR-485-3p could specifically bind to the 3'-untranslated region (3'-UTR) of CtBP1, thereby negatively controlling CtBP1 expression. The overexpression of miR-485-3p in osteosarcoma cells significantly repressed CtBP1 levels and inhibited cell proliferation, colony formation, cell migration and sphere formation. Further analysis indicated that DNA hypermethylation in the promoter region of miR-485-3p caused the downregulation of miR-485-3p. Treatment with the DNA methylation inhibitor 5-aza-2'-deoxycytidine (AZA) resulted in the upregulation of miR-485-3p and the downregulation of CtBP1 as well as inhibited osteosarcoma cell growth. This study provides evidence that CtBP1 is also overexpressed in osteosarcoma cells and demonstrates the underlying mechanism regarding its overexpression. Thus, therapeutically targeting CtBP1 may represent an effective strategy for osteosarcoma therapy.
The misregulation of transforming growth factor-β (TGF-β) signaling can cause tumorigenesis, but the activation and suppression of TGF-β signaling are complicated biological processes. We discovered a protein tyrosine phosphatase nonreceptor 3 (PTPN3)-dependent regulatory mechanism in uncancerous osteoblast cells that involved a stabilizing PTPN3 interaction with the TGF-β type I receptor (TGFBR1), which impaired TGFBR1 ubiquitination by the Smurf2 (Smad ubiquitination regulatory factor 2) E3 ligase. TGFBR1 facilitated the phosphorylation of Smad2/3 (SMAD family member 2 and 3), and the phosphorylated Smad2/3 recruited Smad4 to assemble a complex that then was translocated into the nucleus to initiate gene transcription. By contrast, PTPN3 was significantly downregulated in osteosarcoma cells because the increased DNMT1 (DNA methyltransferase 1) caused a higher methylation level in the promoter of PTPN3. The decrease of PTPN3 failed to stabilize TGFBR1, causing the ubiquitination and degradation of TGFBR1 by Smurf2. The degradation of TGFBR1 impaired the phosphorylation of Smad2/3 and prevented the nuclear translocation of the Smad2/3/4 complex, thereby causing the dysregulation of TGF-β target genes and triggering tumorigenesis. Collectively, our results reveal that the DNA methylation-mediated downregulation of PTPN3 disables the stabilization of TGFBR1 and that the degradation of TGFBR1 by Smurf2 E3 ligase blocks Smad2/3/4-mediated gene expression that promotes the tumorigenesis of osteosarcoma.
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